Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Abstract

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories. Keywords: Sperm cryopreservation. Egg yolk. Sperm analysis. Perivitelline membrane. Canine. ResumoDurante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convenci...

Short‐term preservation of canine sperm‐binding ability and other metrics using the INRA‐96 in comparison to Tris‐egg yolk extender

“…As observed, roughly 90% of the dog spermatozoa were still capable of binding to the perivitelline membrane of egg yolk when compared to fresh samples. Moreover, the values associated with the number of bound spermatozoa are even greater than those previously reported when the assay was first validated for fresh canine semen (Brito et al, 2017).…”

“…As stated by Takeuchi et al (1999), the glycoproteins of the perivitelline membrane of chicken egg yolk are homologous to those of the zona pellucida of certain mammalian species. Thus, with the membrane binding test standardized by Brito et al (2017), it is possible to estimate the fertilizing potential of canine spermatozoa. Based on the results of our study, the INRA‐96 extender provided preservation of the canine sperm‐binding ability better than the Tris‐egg yolk extender after 48 h storage under refrigeration.…”

“…Sperm binding was assessed as described by Brito et al (2017), using perivitelline membranes from fresh, unfertilized hen eggs. The perivitelline membrane was prepared by separating the egg yolk from the albumen, removing the excess with parafilm.…”

“…As stated by Takeuchi et al (1999), the glycoproteins of the perivitelline membrane of chicken egg yolk are homologous to those of the zona pellucida of certain mammalian species. Thus, with the membrane binding test standardized by Brito et al (2017), it is possible to estimate the fertilizing potential of canine spermatozoa.…”

“…Given the orthologous similarity between glycoproteins from the zona pellucida of mammalian oocytes and proteins from the chicken egg perivitelline membrane (Takeuchi et al, 1999), the sperm binding to this subtract arises as an alternative test for sperm functional evaluation, as it permits an indirect and accurate analysis of the ejaculate fertilizing capacity (Campos et al, 2017). Once previously validated for dogs, this test would be an adequate option for testing the extenders' capacity to preserve canine sperm‐binding ability (Brito et al, 2017), in addition to the classical analysis of sperm motility, morphology and membrane integrity.…”